Ubiquitin is a small eukaryotic protein that is synthesized naturally as one of several fusion proteins, which are processed by ubiquitin-specific proteases to release free ubiquitin. The expression of heterologous proteins as fusions to ubiquitin in either prokaryotic or eukaryotic hosts often dramatically enhances their yield, and allows the exposure of any amino acid following cleavage of ubiquitin. The single exception is when proline is the amino acid immediately following ubiquitin; the

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... iquitin-proline bond is poorly cleaved by presently studied ubiquitin-specific proteases. We show that the mouse ubiquitin-specific protease Unp, and its human homolog Unph, can efficiently cleave the ubiquitin-proline bond in ubiquitin fusion proteins of different sizes. N-terminal sequencing of the cleavage products reveals that cleavage occurs precisely at the ubiquitin-proline junction. The biological significance of this cleavage activity is unclear, as ubiquitin-proline fusions do not occur naturally. However, it may indicate a different catalytic mechanism for these ubiquitin-specific proteases and/or that they can cleave ubiquitin-like proteins. Unp and Unph thus represent versatile ubiquitin-specific proteases for cleaving ubiquitin-fusion proteins in biotechnology and basic research, regardless of both the amino acid immediately following ubiquitin, and the size of the fusion partner. Ubiquitin is a highly conserved eukaryotic protein that is invariably synthesized as a fusion protein either to itself or to one of two ribosomal proteins (1). One consequence of this fusion structure is that the action of an endopeptidase is required to cleave the fusion precursors to release free ubiquitin for its conjugation to other proteins as a marker for selective protein degradation (reviewed in Refs. 2 and 3 ). This cleavage is accomplished by members of a large family of enzymes variably termed ubiquitin-specific proteases (Ubps), 1 ubiquitin Cterminal hydrolases, or deubiquitinating enzymes (4 -7).